Phospholipids provide the matrix of all biological membranes, and their metabolic degradation, transportation and participation in membrane remodeling occur.in micelles. In order to elucidate the mechanisms of these processes and to facilitate the use of phospholipid containing micelles as reaction media their structure and physical properties must be delineated. The long term goal of the proposed research is to develop wellcharacterized micellar substrates for the study of lipolytic enzymes. Phospholipases are involved in metabolic degradation of phospholipids;they also participate in vitally important physiological processes including signal transduction, platelet aggregation, cardiac contraction and excitation, and prostaglandin biosynthesis. In these events water soluble lipolytic enzymes act on water insoluble substrates that are organized in supramolecular aggregates (e.g., mixed micelles). Elucidation of the physicochemical properties of the lipid-water interface and its effect on phospholipase catalysis will advance the level of understanding the mechanism of action of these enzymes. The working hypothesis is that by delineating the contributions of the micellar components to the physicochemical properties of the lipid-water interface one will be able to design micelles with targeted physicochemical properties. To test this hypothesis a newly developed spin exchange EPR technique will be used in conjunction with time-resolved fluorescence quenching measurements. 1) Site directed spin labelings, and fluorescent reporter groups will be used to determine the contribution of the alkyl chain to the properties of the micelle, 2) the physicochemical parameters of the polar shell will be determined using the spin probe di-tert.-butyl nitroxide (DTBN), 3) inter-phase solute exchange between the micelles and water will be measured by spin label partitioning, and 4) the relationship between the properties of the lipid-water interface and the rate of phospholipid hydrolysis will be established using a series of secretory phospholipase enzymes. Should the new EPR method prove to be successful in characterizing the mixed micelles here studied, it will open the possibility to apply it to other more complicated supramolecular assemblies (e.g. phospholipids-bile salt mixed micelles) and may become a general technique to characterize micellar aggregates. Relevance to Public Health: Micelles play an important role in biological processes, including drug delivery and membrane function. Elucidation of their structure and properties will provide useful information toward better understanding of membrane-dependent normal and pathological cell functions including cell signaling, inflammation, allergy, apoptosis, as well as membrane fusion.